Previous studies have identified minichromosome maintenance proteins (MCM) as key regulators in the cell cycling process of epithelial tissue (see WO99/21014 and Gonzalez et al; Nature Reviews/Cancer, Vol 5: pp 135-141, February 2005). Multiple conserved mechanisms limit DNA replication to once per cell cycle. An essential role in proliferation for MCMs and their regulators makes them potentially important biomarkers for routine clinical use in cancer detection and prognosis.
MCMs were identified as useful biomarkers of “cell cycle state”, i.e. whether a cell is capable of proliferating rather than being quiescent or senescent. Expression of all 6 MCMs (MCM 2-7) is seen throughout all phases of the cell cycle and is down regulated following exit from the cell cycle into quiescence, differentiation or senescence. This is evident on immunohistochemical examination of normal stratified epithelium, covering a number of tissues including cervix, bladder, colon and oropharynx among other epithelial tissues. In each of these cases, MCMs are confined to the basal proliferative compartment and are absent in the terminally differentiated superficial keratinocytes. By contrast, in pre-invasive epithelial lesions, the proliferative compartment progressively expands with increasing histological grade and this is paralleled by the appearance of MCM positive cells at the surface of the epithelium (more than 90% in high grade squamous epithelial lesions in for example the cervix and approximately 40% in low grade squamous intraepithelial lesions in the same tissue).
The ability of MCMs to distinguish cycling cells from quiescent cells prompted a potential clinical application in cancer screening approaches that relies on the detection of malignant or pre-malignant cells exfoliated from surface epithelia such as in cervical screening or indeed in bladder screening using cell detection techniques from the urine of patients affected by Transitional Cell Carcinoma of the bladder.
The present invention concerns a screen for the detection of prostate cancer. Although MCMs can be used as targets in the detection of malignant cells in a body fluid such as urine, they are not tissue specific. There is, therefore, a need for a means of specifically detecting prostate cancer cells in urine. The present invention is based on the determination that prostate epithelial cells can be detected in urine which can in turn be analysed for malignancy.
Thus according to a first aspect of the present invention there is provided a method for detecting or determining the presence of prostate cancer cells in a sample of body fluid from a subject comprising:
(i) isolating cells from said sample to provide a cell sample;
(ii) contacting said cell sample with a specific binding member capable of binding a prostate antigen; and/or
(iii) contacting said cell sample with a specific binding member capable of binding a minichromosome maintenance (MCM) polypeptide(s); and
(iv) determining the binding of said specific binding member(s) to the cell sample.
Typically the body fluid is not blood or cerebrospinal fluid. The body fluid may be urine or semen. Alternatively the body fluid may be faeces. Preferably the body fluid is urine. Preferably the subject is human.
Cells may be isolated from the body fluid sample by any means known to the skilled person. Typically the cells are isolated by either centrifugation or filtration of the body fluid sample. Preferably the cells are isolated by filtration of the body fluid sample. In a preferred method of the invention the sample is subject to antigen retrieval. Antigen retrieval is standard in the art (see Hiraiwa et al refer to Shin et al (1991) Lab. Invest. 64, 693-702 which provides an exemplary approach). Antigen retrieval conditions may include contacting the cell sample with pH7.8 EDTA buffer at 95° for 45 min in water bath or Microwave.
In a method of the invention the prostate antigen may be an antigen specific for prostate tissue. The prostate antigen may be present in normal (i.e. non-cancerous) and in prostate tumour cells. Examples of prostate antigens include, but are not limited to, Prostatic Acid Phosphatase (PSAP), Prostate Specific Antigen (PSA), Prostate Specific G Protein Coupled Receptor (PSGR), and a-Methylacyl-coenzyme-A racemase (AMACR). Preferably the prostate antigen is PSAP or PSGR. In one embodiment of the invention the prostate antigen is PSA.
In a method of the invention the MCM is selected from the group consisting of MCM 2, 3, 4, 5, 6 and 7. The MCM may be a combination of two or more different MCMs, for example, two different MCMs selected from the group consisting of MCM 2, 3, 4, 5, 6 and 7. For example the MCM may include MCM2 and one other MCM selected from MCM 3, 4, 5, 6 and 7. By way of further example the MCM may include MCM5 and one other MCM selected from MCM 2, 3, 4, 6 and 7. In a preferred method of the invention, the MCM is selected from the group consisting of MCM 2, 3, 5 and 7. In a further preferred method of the invention, the MCM is selected from the group consisting of MCM 2, 5 and 7.
In a method of the invention the MCM may include MOM 2 and MCM 5. In a further method of the invention the MCM may include MCM 2 and MCM 7. In a yet further method of the invention the MCM may include MCM 5 and MCM 7.
As used herein, a “specific binding member” is a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, which may be a protrusion or cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other.
Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate, DNA-DNA (e.g. oligonucleotide). The present invention is generally concerned with antigen-antibody type reactions, although it also concerns small molecules which bind to the antigen defined herein.
The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal.
As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic.
Antibodies which are specific for a target of interest, such as MCM or PSA, PSAP, PSGR or AMACR, may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit) with the protein or a fragment thereof or a cell or virus which expresses the protein or fragment. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, for example using binding of antibody to antigen of interest.
An “antigen binding domain” is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region
“Specific” is generally used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s), e.g., has less than about 30%, preferably 20%, 10%, or 1% cross-reactivity with any other molecule.
The specific binding members of the invention will preferably be, in accordance with the present invention, in “isolated” form. Members will generally be free or substantially free of material with which they are naturally associated such as other polypeptides with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo.
Thus the specific binding member of the invention is preferably an antibody, or fragment thereof. Thus, for example in (ii) the specific binding partner member may be an antibody, or fragment thereof, having an antigen binding domain specific for prostate tissue. For example in (iii) the specific binding member may be an antibody, or fragment thereof, having an antigen binding domain specific for MCM.
The antibody may be a polyclonal antibody, monoclonal antibody, single chain antibody or fragment of any of the foregoing. Preferably the specific binding member is a monoclonal antibody. For example in (ii) the specific binding member may be a monoclonal antibody having an antigen binding domain specific for prostate tissue. Monoclonal antibodies specific to PSA (Dako), PSAP (Sigma), PSGR (Abcam) and AMACR (Dako) are known in the art the details of which are included in Attachment 1. For example in (iii) the specific binding member may be a monoclonal antibody having an antigen binding domain specific for MCM. Monoclonal antibodies specific for MCM are known in the art, for example, anti-MCM2 antibody used in the present study has kindly been provided by the Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ.
The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, “Basic Facts about Hybridomas” in Compendium of Immunology V.II ed. by Schwartz, 1981, which are incorporated by reference.
In a method of the invention, the specific binding members of the invention may be labelled with a detectable label, for example a radiolabel such as I or 99Tc, which may be attached to specific binding members of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidise or alkaline phosphatise. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.
The reactivities of a specific binding member such as an antibody on normal and test samples may be determined by any appropriate means. Other labels include fluorochromes, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine. Other labels include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. In the examples described below, alkaline phophatase or horseradish peroxidise have been employed.
In a preferred aspect of the invention the method comprises the steps of:
(i) isolating cells from said sample to provide a cell sample;
(ii) contacting said cell sample with a specific binding member for a prostate antigen; and
(iii) determining the binding of said specific binding member to the cell sample.
In an alternative aspect of the invention the method comprises the steps of:
(i) isolating cells from said sample to provide a cell sample;
(ii) contacting said cell sample with a specific binding member for one or more MCM; and
(iii) determining the binding of said specific binding member to the cell sample.
In a further preferred aspect of the invention the method comprises the steps of:
(i) isolating cells from said sample to provide a cell sample;
(ii) contacting said cell sample with a specific binding member for prostate antigen; and
(iii) contacting said cell sample with a specific binding member, or specific binding members, for a minichromosome maintenance (MCM) polypeptide; and
(iv) determining the binding of said specific binding members to the cell sample.
The step of contacting said cell sample with a specific binding member for a prostate antigen may be carried out separately, sequentially or simultaneously with the step of contacting said cell sample with a specific binding member, or specific binding members, for a Minichromosome maintenance (MCM) polypeptide. In one embodiment of the invention the step of contacting said cell sample with a specific binding member for prostate antigen is carried out simultaneously with the step of contacting said cell sample with a specific binding member, or specific binding members, for a minichromosome maintenance (MCM) polypeptide.
Preferably the step of contacting said cell sample with a specific binding member for prostate antigen is carried out separately and sequentially with the step of contacting said cell sample with a specific binding member, or specific binding members, for minichromosome maintenance (MCM) polypeptide. Thus in a preferred aspect of the invention there is provided a method for detecting or determining the presence of prostate cancer cells in a sample of body fluid from a subject comprising:
(i) isolating cells from said sample to provide a cell sample;
(ii) contacting said cell sample with a specific binding member capable of binding a prostate antigen; followed by
(iii) contacting said cell sample with a specific binding member capable of binding a minichromosome maintenance (MCM) polypeptide(s); and
(iv) determining the binding of said specific binding member(s) to the cell sample.
In a further aspect of the invention there is provided a composition comprising a specific binding member a prostate antigen, for example PSA and/or PSAP, and a specific binding member, or specific binding members, for a minichromosome maintenance (MCM) polypeptide, for example MCM2 and/or MCM5 and/or 7.
A further aspect of the invention provides a kit for use in a method according to the invention said kit comprising a composition according to the invention. One or more other reagents may be included, such as labelling molecules as described herein. Other reagents may include any combination of or all of a blocking agent to decrease non-specific staining, a storage buffer for preserving specific binding member activity during storage, staining buffer and/or washing buffer to be used, for example, during antibody staining, a positive control, a negative control and so on. Positive and negative controls may be used to validate the activity and correct usage of reagents employed in accordance with the invention and which may be provided in a kit. The design and use of controls is standard and well within the routine capabilities of those of ordinary skill in the art. The kit may further comprise instructions for performing the method of the invention.
In a further aspect of the invention there is provided a method for the treatment of prostate cancer in a subject the method comprising:
(i) detecting or determining the presence of prostate cancer cells in a sample of body fluid, for example urine, from said subject according to the method of the first aspect of the invention;
(ii) where prostate cancer cells are detected in said sample, administering to said subject a treatment for prostate cancer.
Another aspect of the present invention provides for a method of categorising prostate tissue as (i) normal or (ii) potentially or actually pre-cancerous or cancerous, dysplastic or neoplastic, the method including determining binding to a body fluid sample of a specific binding member specific binding member capable of binding a prostate antigen; and/or a specific binding member capable of binding minichromosome maintenance (MCM) polypeptide(s). The pattern or degree of binding may be compared with that for a known normal sample and/or a known abnormal sample.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.